Sodium 4-[4-[[2-methyl-4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo]anilino]-3-nitrobenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL reproductive toxicity (male and female): 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 11th to March 23rd, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop ZRT
- Females: nulliparous, non-pregnant females.
- Age at study initiation: males 90 – 92 days; females 99 – 102 days.
- Weight at study initiation: males 353 – 419 g ; females 212 – 248 g. The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: 2 animals of the same sex/cage before mating; 1 male and 1 female/cage during mating. Pregnant females were housed individually, while males were housed 2 animals/cage after mating. Recovery animals: 2 or 3 animals of the same sex/cage.
- Cages: type III polypropylene/polycarbonate; size of 22 x 32 x 19 cm (width x length x height).
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet for rats and mice. Food was changed at weekly intervals.
- Water: ad libitum, tap water. Fresh drinking water was given daily.
- Health: animals were SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study. Animals that failed to exhibit typical 4-5 days cycles were not included in the study if it was feasible.
- Acclimation period: 34 days.

DETAILS OF FOOD AND WATER QUALITY
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

- Doses: 137, 411 and 1370 mg/kg bw/day, corresponding to 100, 300 and 1000 mg/kg bw/day, calculated by the active ingredient content.
- Nominal concentration of formulation: 13.70, 41.10 and 136.99 mg/ml, corresponding to 10, 30 and 100 mg/ml, calculated by the active ingredient content.
- Dosing volume: 10 ml/kg bw.
- Formulation preparation: formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days before the administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. the substance concentrations in the dosing formulations varied in the range between 101 % and 106 % of the nominal values and confirming the proper preparation of the dosing formulations.

Duration of treatment / exposure:

54 – 55 days males
54 – 55 days females; in one case, 71 days

Frequency of treatment:

once daily

Details on study schedule:

Pre-mating period: 14 days, males and females
Mating period: 1-8 days and 23 days for one female; two male animals (no. 404 and 405) were paired again with female animal no. 423 from mating days 15 and 22, respectively.
Post-mating males: 32-40 days
Gestation period: 21-23 days
Lactation period: 13-18 days

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

calculated by the active ingredient content

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

calculated by the active ingredient content

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

calculated by the active ingredient content

No. of animals per sex per dose:

12 x sex x dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were chosen on the basis of the results of a preliminary toxicity screening test conducted on rats, dosed for 14-day oral (by gavage) at 100, 300 or 1000 mg/kg bw/day.

Parental animals: Observations and examinations:

MORTALITY
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time. Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT
All parental animals were weighed with an accuracy of 1 g. Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not valuated statistically. Body weight was measured on day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated.

FOOD CONSUMPTION
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13, and post-mating days 20, 27, 34, 41, 48 and 53 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

CLINICAL PATHOLOGY
Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry.
In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4) from all parent male animals at termination on Day 54 or 55. All samples for T4 determination are stored at approximately -20 °C

HEMATOLOGY
Blood samples for hematology measurements were collected in tubes containing K3EDTA (spray-dried) and tubes were filled up to the final volume (marked on the tubes). Blood were stored at 2-8 °C until analysis (not longer than for 24 hours) by SYSMEX XT-2000iV.
The following parameters were measured in all selected animals and in recovery animals: White Blood Cell (leukocyte) count (WBC), Red Blood Cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (relative volume of erythrocytes - HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) Hemoglobin (MCH), Mean Corpuscular (erythrocyte) Hemoglobin Concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count (i.e. percentage of neutrophil, lymphocyte, eosinophil, monocyte, basophil.

BLOOD COAGULATION
Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation 3.8 %. Tubes were filled up to the final volume (marked on the tubes). Blood were centrifuged at 2500 rpm for 15 minutes within 20 – 30 minutes after the sampling. Supernatant plasma samples were stored at 2-8 °C and measured.
The following blood coagulation parameters were determined in selected animals and in recovery animals: Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

CLINICAL CHEMISTRY
Blood samples collected for clinical chemistry measurements were drawn in tubes Vacuette 2.5 ml Z Serum Sep C/A (no anticoagulant). At least 1.0 ml blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4500 rpm for 15 minutes. Serum samples were stored at 2-8 °C and measured.
The following parameters were measured in all selected animals and in recovery animals: Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Bile acids (BAC), Sodium concentration (Na+), Potassium concentration (K+), Albumin concentration (ALB), Total Protein concentration (TPROT).

DETERMINATION OF SERUM THYROID HORMONE
Blood samples for determination of serum levels of thyroid hormones (T4) were drawn as for clinical chemistry in the morning hours (approximately between 8h and 10h a.m.). Blood samples were collected from animals as follows: from 2-6 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter); from all dams and 3-8 pups per litter on post-partal/ postnatal day 13; from all parent male animals at termination on Days 54 or 55; from non-pregnant female animals on study Day 56.
All samples for T4 determination were stored at -25 to -15 °C until measurement.
Blood samples from the day 13 pups and the adult males were assessed for serum levels of thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups were not performed because there were no relevant changes in the examined samples (based on observations in postnatal day 13 offspring and parental male animals).
The quantitative determination of thyroid hormones (T4) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.

EXAMINATION OF PLACENTAL SIGN
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If the test was negative on day 13, the examination was repeated on day 14 of gestation.

OBSERVATION OF DELIVER PROCESS
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestational day 21 onwards. All observations and any evidence of abnormal deliveries were considered. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if it was feasible. Extra pups were eliminated by a random selection. Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

Oestrous cyclicity (parental animals):

Measure of female’s ability to become pregnant was performed.

Sperm parameters (parental animals):

Measure of male’s ability to produce sperm that can fertilize eggs was performed.

Litter observations:

LITTER ASSESSMENT
Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0), and on days 4 and 13 post-partum with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups).
All litters were checked and recorded daily for the number of viable and dead pups.

BODY WEIGHT
Therefore individual body weight of pups was also determined on postnatal day 4 and the litter weight was calculated for evaluation on postnatal day 4.

BODY EXAMINATION
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight.
The number of nipples/areolae in male pups was counted on postnatal day 13.

CLINICAL PATHOLOGY
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4) as follows:
- from at least two pups per litter on post-natal day 4 (if it was feasible) - pup blood was pooled by litter.
- from all dams and at least two pups per litter at termination on lactation day 13.
All samples for T4 determination are stored at approximately -20 °C

GROSS EXAMINATION OF DEAD PUPS
Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
One male animal at 100 mg/kg bw/day was found dead and subjected to necropsy on Day 55. All other animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® and were subjected to gross necropsy as follows: parental male animals: after the optionally extended post-mating period on Days 54 or 55; non-pregnant females on Day 54.

GROSS NECROPSY
Gross necropsy was performed on each animal.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size.
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Histological examination was also performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in not mated or non-pregnant females and males these females cohabited with in the low and middle dose groups
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1000 mg/kg bw/day).
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Postmortem examinations (offspring):

SACRIFICE
All other animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® and were subjected to gross necropsy as follows: dams on post-partum day 13 or shortly thereafter (Days 54, 55 or 71) offspring on postnatal day 13 or shortly thereafter.

GROSS NECROPSY
Gross necropsy was performed on each animal.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Reproductive indices:

Gestation index. i.e. measure of pregnancy that provides at least one live pup, was calculated.
Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.

Offspring viability indices:

Post-implantation mortality (intrauterine mortality), post-natal mortality, survival Index and sex ratio were calcualted.

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations.
There were no clinical signs in any group, i.e. the parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 300 or 1000 mg/kg bw/day.
Yellowish color of stool was observed in the bedding material of animals administered with 1000 mg/kg bw/day (12/12 male and 12/12 female) from Day 6 up to the termination of the study. Yellowish color of the faeces was due to the elimination of test item or its metabolite by the gastrointestinal tract.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male animal at 100 mg/kg bw/day was found dead on the day of the scheduled necropsy on Day 55. There were no clinical signs but significant body weight loss was detected during the preceding week. Necropsy observation revealed yellowish fluid in the thoracic cavity and empty intestines indicative of para-gastric treatment.
There was no test item related mortality at 300 or 1000 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was not adversely affected by test item in survival male or female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period. The mean body weight was similar in the control and at 100, 300 and 1000 mg/kg bw/day groups in male animals.
Only in the one male animal (No. 206) died after necropsy and dosed at 300 mg/kg bw/day, significant body weight loss was detected during the preceding week.
Although the mean body weight gain was slightly reduced in male animals at 1000 mg/kg bw/day (days 0-7 and for study overall), these slight change did not result in significant changes in the mean body weight of this groups during the treatment period. Therefore, the mean body weight gain was below the control group with statistical significance in male animals at 1000 mg/kg bw/day for study overall (between Days 0 and 53). These changes in body weight gain were with small degree and not consistent, moreover, there were no significant changes in the mean body weight, therefore minor changes in body weight gain of male animals were considered to be toxicologically not relevant.
Statistical significance with respect to the control was detected in male animals at 300 mg/kg bw/day at the slightly lower mean body weight gain of on Days 20-27 and at the slightly higher mean body weight gain between Days 34-41 and days 41 and 48..
The mean body weight gain was lower than in the control group in female animals at 100 and 1000 mg/kg bw/day during week 1 (between Days 0 and 7) of the pre-mating period. These slight changes were not related to doses and did not cause significant alterations in the mean body weight therefore were judged to be toxicologically not significant.
In the female animals, the body weight development was undisturbed during the gestation and lactation periods. There were no significant differences between the control and test item treated groups in the mean body weight or mean body weight gain (100, 300 and 1000 mg/kg bw/day).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item or treatment related changes in the mean daily food consumption of male animals at 100, 300 or 1000 mg/kg bw/day.
The food consumption was comparable in the control and test item treated animals, i.e. statistically or biologically significant differences were not seen in the mean daily food consumption of male or female animals at any dose level (100, 300 or 1000 mg/kg bw/day) with respect to their control group during the entire observation period (pre-mating and post mating periods in male animals; pre-mating, gestation and lactation periods in female animals).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day.
Slight but statistically significant differences with respect to their controls were noted for some parameters as follows: lower mean corpuscular hemoglobin concentration (MCHC) at 1000 mg/kg bw/day and lower mean platelet count (PLT) at 300 mg/kg bw/day, both in male animals; higher mean percentage of eosinophil granulocytes (EOS) in female animals at 300 mg/kg bw/day.
These differences with respect to control were judged to be toxicologically not relevant due to the minor degree (PLT, EOS and MCHC) and in the lack of dose relevance (PLT, EOS).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
Slight but statistically significant differences were observed between the control and treated groups of male animals at the higher mean concentration of total bilirubin (TBIL; at 1000 mg/kg bw/day) and at the higher mean concentration of sodium (Na+; at 100, 300 and 1000 mg/kg bw/day) as well as albumin (ALB; at 300 mg/kg bw/day).
Additional sporadic observations in males were attributed to biological variation and not related to the test item; they are not statistically significant and values are well within the historical control ranges
In the female animals, statistically significant difference with respect to the control was observed at the slightly mean cholesterol concentrations (CHOL) at 300 and 1000 mg/kg bw/day.
These slight, but statistically significant differences with respect to their controls were considered to have little or no toxicological importance because all these changes were with low degree, values – mean and individual – remained within the historical control ranges (TBIL, Na+, ALB, CHOL) or there were no dose relevance (Na+, ALB, CHOL).
Additional sporadic observations in females were attributed to biological variation and not related to the test item; they are not statistically significant and values are well within the historical control ranges.
Statistically significant difference with respect to the control was noted for the slightly lower thyroid hormone (free T4) level in parental animal at 1000 mg/kg bw/day. However this slight difference was considered to be not relevant as all values were within the historical control (2.72 ± 0.39 ng/dL; n=96; min: 1.91 ng/dL; max = 3.86 ng/dL) except for one animal. Moreover there were no histological changes in the thyroid gland tissue of examined animals at 1000 mg/kg bw/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any treatment-related alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, 100, 300 or 1000 mg/kg bw/day groups, on Day 49).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Yellowish color of stool was also detected in the cages of high dose treated male and female animals (5/5 male and 5/5 female) at the functional (cage side) observations.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (1000 mg/kg/bw/day). There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control, 100 and 1000 mg/kg bw/day).
In the male animals belonging to the treated and control groups (12/12 control; 2/2 at 100 mg/kg bw/day; 12/12 at 1000 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 2/2 at 100 mg/kg bw/day; 12/12 at 1000 mg/kg bw/day), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.

In animals selected for full histological examinations, minimal alveolar emphysema in the lungs (2/5 control male, 1/5 male at 1000 mg/kg bw/day) and acute hemorrhage in the thymus (1/5 male at 1000 mg/kg bw/day) occurred sporadically and these were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.

Hyperplasia of bronchus associated lymphoid tissue (BALT) was observed in single male animals of control (1/5) and 1000 mg/kg bw/day (1/5) groups. Hyperplasia of BALT is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Chronic hematoma surrounded by fibrotic encapsulation was detected in the liver of one dam (1/5) at 1000 mg/kg bw/day, which was considered to be an individual lesion probably due to mechanical injury, attributable to para-gastric treatment.
Involution of the thymus is a normal physiological phenomenon in experimental rats with this age. Regarding the low incidence (1/5 male at 1000 mg/kg bw/day) it was considered to be independent from the treatment.
The histological structure and the cellularity of pituitary with special attention on the cyto-morphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control (5/5 male and 5/5 female) and high dose treated male (5/5) and female (5/5) animals.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver (except hematoma), the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system in surviving animals. The quantity and quality of hemopoietic cells in the bone marrow, and the cyto-morphology of endocrine glands was the same in the control and high dose treated animals.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A test item influence on the estrous cycle was not detected at any dose level (100, 300 or 1000 mg/kg bw/day).
The mean length of estrous cycle of animals at 100 mg/kg bw/day was slightly shorter than in the control group. This minor change was not considered to be related to the treatment, but indicative of biological variation.
There were no differences between the control and treated groups (100, 300 and 1000 mg/kg bw/day) in number or percentage of animals with regular or irregular cycles, in the mean number of cycles, mean number of days in pro-estrous, estrous or diestrus, in number or percentage of animals in prolonged estrous or diestrous during the pre-mating period.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were comparable in the control and in 100, 300 and 1000 mg/kg bw/day groups (male and female).
Statistical significance was observed at the slightly lower copulatory index of male animals at 1000 mg/kg bw/day because one male animal (1/12) failed to mate within 14 days. The fertility index of male animals at 100 mg/kg bw/day was lower than in the control group as two male animals (2/12) did not fertilize their females.
In the female animals at 100 and 1000 mg/kg bw/day, the fertility index was lower than in the control group.
These minor changes in the copulatory or fertility indices (male or female) were considered to be not related to the test item because similar incidences also occur in non-treated experimental ratsof this strain, both males and females, moreover there was no dose relevance in the fertility index.
The copulatory index, percentage of pregnant females, non-pregnant females, and dams delivered, pregnants with liveborn(s) and the pre-coital interval, mean number of conceiving days and gestation indices were comparable in female animals of control and test item treated animals.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were comparable in the control and in 100, 300 and 1000 mg/kg bw/day groups (male and female).
Statistical significance was observed at the slightly lower copulatory index of male animals at 1000 mg/kg bw/day because one male animal (1/12) failed to mate within 14 days. The fertility index of male animals at 100 mg/kg bw/day was lower than in the control group as two male animals (2/12) did not fertilize their females.
In the female animals at 100 and 1000 mg/kg bw/day, the fertility index was lower than in the control group.
These minor changes in the copulatory or fertility indices (male or female) were considered to be not related to the test item because similar incidences also occur in non-treated experimental rats of this strain moreover there was no dose relevance in the fertility index.
The copulatory index, percentage of pregnant females, non-pregnant females, and dams delivered, pregnants with liveborn(s) and the pre-coital interval, mean number of conceiving days and gestation indices were comparable in female animals of control and test item treated animals.

DELIVERY DATA OF DAMS
Effects observed, non-treatment-related.
There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups.
The number of pregnant females and dams delivered, the mean number of implantations, post-implantation loss, duration of pregnancy, and pups births (total, live, still birth, viable) and live birth index were comparable in all groups.
Statistical significance was noted with respect to the control for the lower mean post-implantation loss at 100 and 1000 mg/kg bw/day dams. These deviations had no toxicological meaning and were considered to be indicative of biological variation.

Dose descriptor:

NOAEC

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Test item related significant clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring showing clinical signs (cold, not suckled) was lower at 1000 mg/kg bw/day than in the control group.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices. There were no significant differences between the control and test item treated groups (100, 300 or 1000 mg/kg bw/day) in the mean number of dead offspring (including missing and cannibalized pups) per litter.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The offspring’s body weight development was not adversely affected between postnatal days 0 and 13. The mean body weight of offspring was slightly reduced at 1000 mg/kg bw/day however the difference with respect to the control was less than 10 % therefore it was considered to be toxicologically not relevant.
Statistically significant difference with respect to the control was detected at 1000 mg/kg bw/day in the slightly lower mean pup weight on postnatal days 0 and 13 and at the lower mean male and female pup weights at 300 and 1000 mg/kg bw/day on postnatal day 4.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
One male pup in the control group was subjected to necropsy on postnatal day 0. There were no macroscopic changes in the organs of this pup and the lung flotation test was negative referring to intrauterine death.
One male pup at 300 mg/kg bw/day was partially cannibalized and not suitable for necropsy observations.

SEX DISTRIBUTION
There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0, 4 or 13.

DEVELOPMENT
The anogenital distances (in male or female offspring) or nipple retention (male) were not affected by test item at 100, 300 and 1000 mg/kg bw/day).
The anogenital distance, both absolute and normalized, in male rats of all the treated groups resulted to be consistent with the control.
The anogenital distance normalized was slightly but statistically significantly longer in female pups of the tests item treated dams (300 and 1000 mg/kg bw/day); on the contrary, the absolute anogenital distance resulted to be not impacted.
. In the lack of dose relevance and due to the minor degree, this slight difference with respect to the control was not considered to be toxicologically important.
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on postnatal day 13.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

NOAEL reproductive toxicity (male and female): 1000 mg/kg bw/day
NOAEL developmental toxiciy: 1000 mg/kg bw/day

Executive summary:

The objective of the study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage), according to OECD 422.

Test item was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of rats consisting of 12 animals per sex per group.

Test substance concentrations in the dosing formulations varied in the range between 101 % and 106 % of the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 54 or 55 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 19, i.e. up to the day before necropsy (altogether for 54 or 55 days; one female animal at 1000 mg/kg bw/day was administered for 71 days due to the late mating).

There was no test item related mortality at any dose level; one male animal at 100 mg/kg bw/day was found dead on the day of scheduled necropsy.

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. The behaviour and physical condition of the animals was not impaired at each dose level during the entire treatment period. Body weight or body weight gain were not affected.

A test item influence on the estrous cycle was not detected at any dose level, as well as no test item related differences in comparison with the control in the examined parameters of reproductive performance or in the delivery parameters of dams was recorded.

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at none of tested doses. There were no test item related adverse effects on the examined clinical chemistry parameters in both male and female.

Necropsy did not revealed any effect in male and female animals; furthermore, there were no test item related changes in the weights of brain, testes and epididymides of male animals at any dose level.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes.

No adverse effect on the mortality, clinical signs or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected. The offspring’s body weight development was slightly depressed at 1000 mg/kg bw/day between postnatal days 0 and 13 but the difference with respect to the control was not considered toxicologically relevant due to its minor degree (<10 %).

 

Conclusion

Under the conditions, the test item administered at 100, 300 or 1000 mg/kg bw/day by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female rats. The development of the F1 offspring was not impaired from conception to birth or from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A screening for reproductive / developmental toxicity test was performed to obtain initial information on the toxic potential of test item and on the possible effects of the test item when repeatedly administered orally (by gavage), according to OECD 422.

Test item was administered orally once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of rats consisting of 12 animals per sex per group. Test substance concentrations in the dosing formulations varied in the range between 101 % and 106 % of the nominal values and confirming the proper preparation of the dosing formulations. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 54 or 55 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 19, i.e. up to the day before necropsy (altogether for 54 or 55 days; one female animal at 1000 mg/kg bw/day was administered for 71 days due to the late mating).

There was no test item related mortality at any dose level; one male animal at 100 mg/kg bw/day was found dead on the day of scheduled necropsy. Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. The behaviour and physical condition of the animals was not impaired at each dose level during the entire treatment period. Body weight or body weight gain were not affected.

A test item influence on the estrous cycle was not detected at any dose level, as well as no test item related differences in comparison with the control in the examined parameters of reproductive performance or in the delivery parameters of dams was recorded.

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at none of tested doses. There were no test item related adverse effects on the examined clinical chemistry parameters in both male and female.

Necropsy did not revealed any effect in male and female animals; furthermore, there were no test item related changes in the weights of brain, testes and epididymides of male animals at any dose level.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes.

No adverse effect on the mortality, clinical signs or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected. The offspring’s body weight development was slightly depressed at 1000 mg/kg bw/day between postnatal days 0 and 13 but the difference with respect to the control was not considered toxicologically relevant due to its minor degree (<10 %).

Under the conditions, the test item administered at 100, 300 or 1000 mg/kg bw/day by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female rats. The NOAEL for systemic toxicity of male/female rats was indicated ot be 1000 mg/kg bw/day and the NOAEL for reproductive performance of male/female rats was indicated to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

NOAEL developmental toxiciy: 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Ascreening for reproductive / developmental toxicity test was performed to obtain initial information on the toxic potential of test item and on the possible effects of the test item when repeatedly administered orally (by gavage), according to OECD 422.

Test item was administered orally once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of rats consisting of 12 animals per sex per group. Test substance concentrations in the dosing formulations varied in the range between 101 % and 106 % of the nominal values and confirming the proper preparation of the dosing formulations. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 54 or 55 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 19, i.e. up to the day before necropsy (altogether for 54 or 55 days; one female animal at 1000 mg/kg bw/day was administered for 71 days due to the late mating).

There was no test item related mortality at any dose level; one male animal at 100 mg/kg bw/day was found dead on the day of scheduled necropsy. Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. The behaviour and physical condition of the animals was not impaired at each dose level during the entire treatment period. Body weight or body weight gain were not affected.

A test item influence on the estrous cycle was not detected at any dose level, as well as no test item related differences in comparison with the control in the examined parameters of reproductive performance or in the delivery parameters of dams was recorded.

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at none of tested doses. There were no test item related adverse effects on the examined clinical chemistry parameters in both male and female.

Necropsy did not revealed any effect in male and female animals; furthermore, there were no test item related changes in the weights of brain, testes and epididymides of male animals at any dose level.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes.

No adverse effect on the mortality, clinical signs or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected. The offspring’s body weight development was slightly depressed at 1000 mg/kg bw/day between postnatal days 0 and 13 but the difference with respect to the control was not considered toxicologically relevant due to its minor degree (<10 %).

The development of the F1 offspring was not impaired from conception to birth or from birth to post-natal day 13 at any dose level after repeated oral administration of dams; thus, the NOAEL for F1 Offspring was established to be 1000 mg/kg bw/day.

Justification for classification or non-classification

According to CLP Regulation (EC) No 1272/2008, 3.7 Reproductive toxicity section, reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

 

Based on the available information, the substance does not meet the criteria to be classified for reproductive toxicity, according to CLP Regulation (EC) 1272/2008.